Assaying protein fragments in body fluids

ABSTRACT

Degradation of body proteins such as bone collagen is studied by detecting in body fluids isomerized peptide fragments of said proteins in which peptide bonding to the α-carboxylic acid group of aspartic acid or asparagine has become isomerized to an amide bond to the side chain carboxylic acid group of aspartic acid. Antibodies specific for the isomerized peptide sequences are used in assays.

This application is a national stage entry of EP96/01228.

The present invention relates to the assaying of collagen or otherprotein degradation products and materials useful therefor.

COLLAGENS AND DISORDERS OF COLLAGEN METABOLISM

Osteoporosis is the most common bone disease in humans. Primaryosteoporosis, accompanied by increased susceptibility to fractures,results from a progressive reduction in skeletal bone mass. It isestimated to affect 15-20 million individuals in the USA alone. Itsbasis is an age-dependant imbalance in bone remodelling, i.e. in therates of formation and resorption of bone tissue.

In the USA about 1.2 million osteoporosis-related fractures occur in theelderly each year including about 538,000 compression fractures of thespine, about 227,000 hip fractures and a substantial number of earlyfractured peripheral bones. Between 12 and 20% of the hip fractures arefatal because they cause severe trauma and bleeding, and half of thesurviving patients require nursing home care. Total costs fromosteoporosis-related injuries now amount to at least $10 billionannually in the USA (Riggs, New England Journal of Medicine, 327:620-627(1992)).

Osteoporosis is most common in postmenopausal women who, on average,lose 15% of their bone mass in the 10 years after menopause. Thisdisease also occurs in men as they get older and in young amenorrheicwomen athletes. Despite the major, and growing, social and economicconsequences of osteoporosis, the availability of reliable assays formeasuring bone resorption rates in patients or in healthy subjects isvery limited. Other disorders entailing (and correlated with)abnormalities in collagen metabolism include Paget's disease, Marfan'ssyndrome, osteogenesis imperfecta, neoplastic growth in collagenoustissue, dwarfism, rheumatoid arthritis, osteoarthritis and vasculitissyndrome.

Three known classes of human collagen have been described to date. TheClass I collagens, subdivided into types I, II, III, V, and XI, areknown to form fibrils. The amino-acid sequence of type I-III (to theextent it has been elucidated) is given in Appendix A of WO95/08115.

Collagen type I accounts for more than 90% of the organic matrix ofbone. Therefore, in principle, it is possible to estimate the rate ofbone resorption by monitoring the degradation of collagen type I.Likewise, a number of other disease states involving connective tissuecan be monitored by determining the degradation of collagen. Examplesare collagen type II degradation associated with rheumatoid arthritisand osteoarthritis and collagen type III degradation in vasculitissyndrome.

Amino acid sequences of human type III collagen, human pro α1(II)collagen, and the entire prepro α1(III) chain of human type III collagenand corresponding cDNA clones have been investigated and determined byseveral groups of researchers; see Loil et al., Nucleic Acid Research12:9383-9394 (1984): Sangiorgi et al., Nucleic Acids Research,13:2207-2225 (1985); Baldwin et al., Biochem J., 262:521-528 (1989); andAla-Kokko et al., Biochem. J., 260:509-516 (1989).

Type I, II, and III collagens are all formed in the organism asprocollagen molecules, comprising N-terminal and C-terminal propeptidesequences, which are attached to the core collagen molecules. Afterremoval of the propeptides, which occurs naturally in vivo duringcollagen synthesis, the remaining core of the collagen moleculesconsists largely of a triple-helical domain having terminal telopeptidesequences which are non-triple-helical. These telopeptide sequences havean important function as sites of intermolecular cross-linking ofcollagen fibrils extracellularly. The alpha-helical region also includescrosslinkable sites.

Intermolecular cross-links provide collagen fibrils with biomechanicalstability. The formation of these cross-links is initiated bymodification of lysine and hydroxylysine residues to the correspondingaldehydes. Several of these residues located on adjacent chains ofcollagen will spontaneously form different intermolecular cross-links.The exact position of the sites for cross-linking on collagentelopeptides and from the helical region has been previously described.See, for example, Kuhn, K., in Immunochemistry of the extracellularmatrix, 1:1-29, CRC Press, Inc., Boca Raton, Fla. (1982), Eyre, D. R.,Ann. Rev. Biochem., 53:717-48 (1984) or U.S. Pat. Nos. 5,140,103 and5,455,179. Furthermore, the amino acid sequences of some potential sitesfor cross-linking in type I, II, and III collagen are given in Table 1below.

The fibrous proteins, collagen and elastin, are cross-linked by a uniquemechanism based on aldehyde formation from lysine or hydroxylysine sidechains. Four homologous loci of cross-linking are evident in moleculesof type I, II and III collagens (for review see Kuhn, K., inImmunochemistry of the extracellular matrix, 1:1-29 (1982)). Two arealdehyde sizes, one in each telopeptide region. The other two sites arehydroxylysine symmetrically placed at about 90 residues from each end ofthe molecule. When collagen molecules pack into fibrils, these lattersites in the helical region align and react with telopeptide aldehydesin adjacent molecules. There is now strong evidence that3-hydroxypyridinium residues are the mature cross-link coming fromhydroxylysine-derived aldehydes. The mature cross-linking residues ofthe other pathway, i.e. from aldehyde formation of lysine residues, arehowever, still unknown.

As illustrated by formula in EP-0394296 discussed below, the two3-hydroxypyridinium cross-links have been found to be hydroxylysylpyridinoline (also known simply as "pyridinoline") and lysylpyridinoline (also known as "deoxypyridinoline"). These cross-linkingcompounds are naturally fluorescent. Some hydroxylysyl pyridinolinecross-link are found to be glycosylated as discussed for instance inEP-A-0424428.

However, as described in Last et al, Int. J. Biochem. Vol. 22, No. 6, pp559-564, 1990 other crosslinks occur naturally in collagen.

PRIOR ART ASSAYS FOR COLLAGEN DEGRADATION

In the past, assays have been developed for monitoring degradation ofcollagen in vivo by measuring various bio-chemical markers, some ofwhich have been degradation products of collagen.

For example, hydroxyproline, an amino acid largely restricted tocollagen, and the principal structural protein in bone and all otherconnective tissues, is excreted in urine. Its excretion rate is known tobe increased in certain conditions, notably Paget's disease, a metabolicbone disorder in which bone turnover is greatly increased, as discussedfurther below.

For this reason, urinary hydroxyproline has been used extensively as anamino acid marker for collagen degradation; Singer, F. R. et al.,Metabolic Bone Disease, Vol. II (eds. Avioli, L. V., and Kane, S. M.),489-575 (1978), Academic Press, New York.

U.S. Pat. No. 3,600,132 discloses a process for the determination ofhydroxyproline in body fluids such as serum, urine, lumbar fluid andother intercellular fluids in order to monitor deviations in collagenmetabolism. The patent states that hydroxyproline correlates withincreased collagen anabolism or catabolism associated with pathologicalconditions such as Paget's disease, Marfan's syndrome, osteogenesisimperfecta, neoplastic growth in collagen tissues and in various formsof dwarfism.

Bone resorption associated with Paget's disease has also been monitoredby measuring small peptides containing hydroxyproline, which areexcreted in the urine following degradation of bone collagen; Russell etal., Metab. Bone Dis. and Rel. Res. 4 and 5, 2250262 (1981), and Singer,F. R., et al., supra.

In the case of Paget's disease, the increased urinary hydroxyprolineprobably comes largely from bone degradation; hydroxyproline, however,generally cannot be used as a specific index for bone degradation. Muchof the hydroxyproline in urine may come from new collagen synthesis(considerable amounts of the newly made protein are degraded andexcreted without ever becoming incorporated into tissue fabric), andfrom turnover of certain blood proteins as well as other proteins thatcontain hydroxyproline.

Furthermore, about 80% of the free hydroxyproline derived from proteindegradation is metabolised in the liver and never appears in the urine.Kiviriko, K. I., Int. Rev. Connect. Tissue Res. 5:93 (1970), and Weiss,P. H. and Klein, L., J. Clin. Invest. 48:1 (1969). Hydroxyproline is agood marker for osteoporosis as it is specific for collagen in boneseven if it is not specific for bone resorption, but it is troublesome tohandle.

Hydroxylysine and its glycoside derivatives, both peculiar tocollagenous proteins, have been considered to be more accurate thanhydroxyproline as markers of collagen degradation. However, for the samereasons described above for hydroxyproline, hydroxylysine and itsglycosides are probably equally non-specific markers of bone resorption;Krane, S. M. and Simon, L. S., Develop. Biochem. 22:185 (1981).

Other researchers have measured the cross-linking compound3-hydroxypyridinium in urine as an index of collagen degradation injoint diseases. See, for back-ground and as examples, Wu and Eyre,Biochemistry, 23:1850 (1984): Black et al., Annals of the RheumaticDiseases, 45:969-973 (1986); and Seibel et al., The Journal ofDermatology, 16:964 (1989). In contrast to the present invention, theseprior researchers have hydrolysed peptides from body fluids and thenlooked for the presence of free 3-hydroxypyridinium residues.

Assays for determination of the degradation of type I, II, and IIIcollagen are disclosed in EP-0394296 and U.S. Pat. No. 4,973,666 andU.S. Pat. No. 5,140,103. However, these patents are restricted tocollagen fragments containing the cross-linker 3-hydroxypyridinium.Furthermore, the above mentioned assays require tedious and complicatedpurifications from urine of collagen fragments containing3-hydroxypyridinium to be used for the production of antibodies and forantigens in the assays.

At present very few clinical data using the approach described in U.S.Pat. No. 4,973,666 and U.S. Pat. No. 5,140,103 are available.Particularly, no data concerning the correlation between the urinaryconcentration (as determined by methods described in the above mentionedpatents) of 3-hydroxypyridinium containing telopeptides of type Icollagen and the actual bone loss (as determined by repeatedmeasurements by bone densiometry) have been published. The presence of3-hydroxypyridinium containing telopeptides in urine requires the properformation in bone tissue of this specific cross-linking structure atvarious times before the bone resorbing process. Very little informationon these processes is available and it would be desirable to avoid thisdependence of the correct formation of the cross-linking structure.

GB Patent Application No. 2205643 reports that the degradation of typeIII collagen in the body can be quantitatively determined by measuringthe concentration of an N-terminal telopeptide from type III collagen ina body fluid. This method uses antibodies generated to N-terminaltelopeptides released by bacterial collagenase degradation of type IIIcollagen, said telopeptides being labelled and used in the assay.

Schroter-Kermani et al., Immunol. Invest. 19:475-491 (1990) describeimmunological measurement systems based on CNBr fragments of collagentype I and II. Use is made of pepsin-solubilised collagen, leaving thetelopeptides in the tissue (cf. the above mentioned GB PatentApplication No. 2205643). There is therefore no conformity between thefragments and the antibodies raised therefrom. Further, the referenceonly describes measurements on extracted tissue samples.

The development of a monoclonal antibody raised againstpepsin-solubilised type I collagen is described in Werkmeister et al.,Eur. J. Biochem. 1987:439-443 (1990). The antibody is used forimmunohistochemical staining of tissue segments and for measuring thecollagen content in cell cultures. The measurements are not carried outon body fluids.

EP Patent Application No. 0505210 describes the development of antibodyreagents by immunisation with purified cross-linked C-terminaltelopeptides from type I collagen. The immunogen is prepared bysolubilising human bone collagen with bacterial collagenase. Theantibodies thus prepared are able to react with both cross-linked andnon-cross-linked telopeptides, and cross-linkers other thanpyridinoline.

International Patent Application No. WO 91/09114 discloses certainsynthetic peptides which are used to promote cellular adhesion to asolid substrate. The use of the synthetic peptides as immunologicalreagents is not mentioned.

There are a number of reports indicating that collagen degradation canbe measured by quantitating certain procollagen peptides. Propeptidesare distinguished from telopeptides and alpha-helical region of thecollagen core by their location in the procollagen molecule and thetiming of their cleavage in vivo; see U.S. Pat. No. 4,504,587; U.S. Pat.No. 4,312,853; Pierard et al., Analytical Biochemistry 141:127-136(1984); Niemela, Clin. Chem. 31/8:1301-1304 (1985); and Rohde et al.,European Journal of Clinical Investigation, 9:451-459 (1979).

EP Patent Application No. 0298210 and No. 0339443 both describeimmunological determination of procollagen peptide type III andfragments thereof. Further, a method based on the measurement ofprocollagen is disclosed in EP Patent Application No. 0465104.

The use of synthetic peptides with sequences derived from type IXcollagen for the development of immunological reagents is disclosed inPCT Patent Application No. WO90/08195. Likewise the applicationdescribes the use of the antibodies thus produced for the determinationof type IX collagen fragments in body fluids.

U.S. Pat. No. 4,778,768 relates to a method of determining changesoccurring in articular cartilage involving quantifying proteoglycanmonomers or antigenic fragments thereof in a synovial fluid sample.

Dodge, J: Clin Invest 83:647-661 (1981) discloses methods for analysingtype II collagen degradation utilising a polyclonal antiserum thatspecifically reacts with unwound alpha-chains and cyanogenbromide-derived peptides of human and bovine type II collagens. Thedegradation products of collagen were not detected in a body fluid, buthistochemically by staining of cell cultures, i.e. by "in situ"detection.

WO94/03813 describes a competitive immunoassay for detecting collagen orcollagen fragments in a sample wherein a binding partner containing asynthetic linear peptide corresponding to the non-helical C-terminal orN-terminal domain of collagen is incubated with an antibody to thelinear synthetic peptide and the sample, and wherein the binding of theantibody to the binding partner is determined.

WO95/08115 relates to assay methods in which collagen fragments in abody fluid are determined by reaction with an antibody which is reactivewith a synthetic peptide. The assay may be a competition assay in whichthe sample and such a peptide compete for an antibody, possibly apolyclonal antibody raised against fragments of collagen obtained bycollagenase degradation of collagen. Alternatively, it may be an assayin which an antibody, possibly a monoclonal antibody, is used which hasbeen raised against such a synthetic peptide.

One particular peptide fragment which we have found in body fluid,particularly urine, is of the formula (SEQ ID NO:1): ##STR1##

In the above formula, K-K-K represents cross-link which may for instancebe a hydroxypyridinium cross-link but may be any naturally occurringcross-link and specifically any of those discussed in the abovereferenced paper of Last et al.

A larger peptide fragment including the above smaller fragment isreported in EP 0394296.

We have now discovered that a proportion of the "peptide" fragments inbody fluid are related to peptides of equivalent amino acid sequence,e.g. peptides of formula 1, by the isomerization of aspartic acid in theformula to isoaspartic acid. We put "peptides" in quotes here as ofcourse the isomerization means that these species are no longer properlyregarded as being peptides.

The isomerization of proteins containing aspartic acid has been reportedpreviously to be a spontaneous reaction occurring under physiologicalconditions.

See for instance Brennan et al Protein Science 1993, 2, 331-338,Galletti et al, Biochem. J. 1995, 306, 313-325, Lowenson et al, BloodCells 1988, 14, 103-117 and Oliya et al, Pharmaceutical Research, Vol.11, No. 5, 1994, p.751.

The isomerization has the effect of transferring that part of thepeptide chain which runs downstream of the aspartic acid residue in thecarboxy terminus direction from the alpha carboxylic acid of theaspartic acid to which it is bonded via a peptide bond in the normalprotein to the side chain carboxylic acid in a non-peptide amide bond,as shown below: ##STR2## The non-peptide bonded aspartic acid residue istermed "isoaspartic acid".

Similar isomerization can occur in proteins containing asparagineresidues (i.e. with --NH₂ instead of --OH in the starting protein in theabove reaction scheme).

The above discovery indicates that this isomerization also occurs inbone tissue and the extent of isomerization is expected therefore to bemarker for the age of the bone tissue concerned.

Furthermore, the presence amongst such bone peptide fragments of theisomerized peptides provides confirmation that the fragments indeedderive from bone degradation and not some other source such as thedegradation of newly formed collagen never incorporated into bone.

Accordingly, the present invention now provides in a first aspect amethod of measurement of the rate of degradation of a body protein suchas collagen, e.g. from bone, comprising determining the amount of one ormore isoaspartic acid containing species in a body fluid.

The isomerized peptides in question may be characteristic of type I,type II or type III collagen, but preferably are characteristic of typeI collagen.

More preferably, such a method determines the amount of one or morespecific isoaspartic acid containing isomerized peptides present in saidbody fluid.

Preferably, the method determines the amount of the isomerized peptideof formula 2 (below) present in said body fluid (SEQ ID NO:2): ##STR3##wherein one or both of * is isoaspartic acid, or of one or moreisomerized peptides incorporating an epitope cresent in the isomerizedpeptide of formula 2 which contains isoaspartic acid.

In the above formula, K-K-K is a cross-link such as a hydroxypyridiniumcross-link which may be pyridinoline (which may be glycosylated ornon-glycosylated) or deoxypyridinoline.

Preferably, said determination is carried out using an immunologicalbinding partner specific for an isoaspartic acid containing speciespresent in the sample during the procedure, preferably said isomerizedpeptide of formula 2 or a isomerized peptide incorporating an epitopepresent in the isomerized peptide of formula 2 which containsisoaspartic acid.

The immunological binding partner may be a monoclonal or polyclonalantibody. By the requirement that the immunological binding partner bespecific for the isoaspartic acid containing species is meant that theimmunological binding partner distinguishes between said species and theanalogous aspartic acid containing species (peptide) to an extent usefulin the assay.

Suitable immunological binding partners also include fragments ofantibodies capable of binding the same antigenic determinant includingFab, Fab' and F(ab')₂. fragments.

Preferably, the immunological binding partner is an antibody raisedagainst a linear isomerized peptide, preferably a synthetic isomerizedpeptide, corresponding to a sequence within collagen with isoasparticacid substituting in said amino acid sequence for aspartic acid in saidcollagen protein sequence

The assay may take many forms including ELISA, RIA, or IRMA, proceduresfor which are too well known to warrant description here.

In a second aspect, the invention includes the use in an assay forcollagen derived isomerized peptides of a synthetic isomerized peptidehaving an amino acid sequence corresponding to a sequence withincollagen with isoaspartic acid substituting in said amino acid sequencefor aspartic acid in said collagen protein sequence. In a competitionassay, the said synthetic isomerized peptide may be used to compete foran immunological binding partner with one or more isomerized peptides inthe sample.

In an ELISA of this type, the synthetic peptide may be immobilised on asolid support. A sample may be incubated with a polyclonal antibody forthe synthetic isomer of a peptide in contact with the solid support andafter washing, a peroxidase-conjugated (revealing) antibody may beadded. After further incubation, a peroxidase substrate solution isadded. By competition, peptide isomer in the sample reactive with theantibody inhibits the peroxidase reaction.

Alternatively, the synthetic peptide isomer may be used to raise amonoclonal immunological binding partner. The synthetic isomerizedpeptide need not then be a competing agent in the assay. For instance,collagenase treated collagen may be purified and immobilised onto thesolid support and an ELISA may be carried out using a monoclonalantibody.

Accordingly, in a third aspect, the invention includes an antibody,preferably a monoclonal antibody, specific for an amino acid sequencecorresponding to a sequence within a protein, e.g. collagen withisoaspartic acid substituting in said amino acid sequence for asparticacid in said protein, e.g. collagen-sequence.

In a preferred embodiment of this aspect of the invention, the antibodyis specific for an isomerized peptide sequence including the sequenceEKAHiDGGR (SEQ ID NO:3) or containing an epitope specific for thepresence of iD present in said sequence, wherein iD is isoaspartic acid.

Accordingly, this aspect of the invention includes an antibody,preferably a monoclonal antibody reactive with an epitope containing,contained in, or constituted by the peptide isomer sequence EKAHiDGGR(SEQ ID NO:3), wherein iD is isoaspartic acid.

In a fourth aspect, the invention provides an antibody, preferably amonoclonal antibody, raised against a peptide isomer having an aminoacid sequence corresponding to a sequence within a protein, e.g.collagen with isoaspartic acid substituting in said amino acid sequencefor aspartic acid in said collagen protein sequence.

The invention includes cell lines producing monoclonal antibodiesaccording to the third or fourth aspects of the invention.

The invention also includes antibodies according to the third or fourthaspects of the invention coupled to a detectable marker. Suitabledetectable markers include, but are not limited to, enzymes,chromophores, fluorophores, coenzymes, enzyme inhibitors,chemiluminescent materials, paramagnetic materials, spin labels,radio-isotopes, nucleic acid or nucleic acid analogue sequences.

In a fifth aspect, the invention includes the use in an assay forcollagen or other protein derived isomerized peptides of an antibodyspecific for an amino acid sequence corresponding to a sequence withinthe protein, (e.g. collagen) with isoaspartic acid substituting in saidamino acid sequence for aspartic acid in said protein (e.g. collagen)sequence to obtain information regarding the amount of isoaspartic acidcontaining peptide isomer or isomers in said body fluid.

In a sixth aspect, the invention includes a synthetic peptide isomerhaving an amino acid sequence corresponding to a sequence withincollagen with isoaspartic acid substituting in said amino acid sequencefor aspartic acid in said collagen protein sequence, preferably in atleast the substantial absence of the corresponding peptide.

Preferably there is a glycine residue adjacent the aspartic acid residuein the native peptide form of the amino acid sequence, as an adjacentglycine facilitates the isomerization of aspartic acid.

Antibodies may be prepared which are respectively selective for one ormore aspartic acid containing peptides and for their isoaspartic acidcontaining analogues. It is then possible to carry out an assay for bothvariants of the peptide or peptides. The relative amount of isoasparticacid will provide an indication of the age of the protein which is beingbroken down protein and of the bone if the assay is for a type ofcollagen fragment. Accordingly, in a sixth aspect the invention providesa method of obtaining information regarding collagen resorption in apatient, comprising measuring in a body fluid the relative amounts of atleast one aspartic acid containing peptide derived from collagen and acorresponding isoaspartic acid containing peptide analogue.

The invention also includes test kits useful in the methods describedabove. Such kits may comprise an antibody according to the third orfourth aspect of the invention, or similarly specific antibody fragment,preferably in combination with any one or more of:

a synthetic peptide analogue containing isoaspartic acid reactive withthe antibody,

an antibody-enzyme conjugate and/or a substrate therefor,

an enzyme conjugate-substrate reaction stopping composition, or

a wash solution.

The invention may be applied both to humans and to animals.

Suitable body fluids include, human or animal urine, blood, serum,plasma and synovial fluid. It is contemplated that the method may alsobe used e.g. on saliva and sweat. The body fluid may be used as it is,or it may be purified prior to the contacting step. This purificationstep may be accomplished using a number of standard procedures,including, but not limited to, cartridge adsorption and elution,molecular sieve chromatography, dialysis, ion exchange, aluminachromatography, hydroxyapatite chromatography, and combinations thereof.

The invention is described in more detail below. Reference is made tothe appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the HPLC separation of a synthetic peptide and a peptideanalogue of the sequence EKAH*GGR (SEQ ID NO:4) differing at * betweenisoaspartic acid (peak 2) and normal aspartic acid (peak 3) as describedin Example 3a;

FIG. 2 shows the differing reactivity of the separated peptide andpeptide analogue of FIG. 1 in two forms of ELISA, as described inExample 3a; and

FIGS. 3(a) and 3(b) show the fluorescence (a) and absorbance (b) ofcrosslinked peptides and peptide analogues from urine separated by HPLCas described in Example 3(b).

FIGS. 4 and 5 show the results of assays on serum using differentantibodies to monitor the effect of bisphosphonate treatment on boneresorption.

In a preferred embodiment of the method according to the invention, theassaying of type I, II or III collagen fragments in urine or other bodyfluid is performed by an inhibition ELISA (enzyme linked immunosorbentassay) by metering off a sample of urine body fluid contacting thesample with a synthetic peptide analogue having a sequence derived fromcollagen and with an antibody, which is immunoreactive with thesynthetic peptide analogue. The synthetic peptide analogue isimmobilised on a solid support. The antibody may be raised against thesynthetic peptide analogue or may be raised against collagen degradationproducts and screened for by use of such a synthetic peptide analogue.

PREPARATION OF SYNTHETIC PEPTIDE ANALOGUES

The preparation of synthetic peptides and peptide analogues may beperformed according to procedures well known in the art, e.g. bysolid-phase peptide synthesis techniques commonly described as"Merrifield synthesis". Also classical solution phase techniques may beused. Sequences of interest include potential sites for collagencross-linking (see for example Kuhn, K., in Immunochemistry of theextracellular matrix, 1:1-29(1982), Eyre, D. R., Ann. Rev.Biochem.53:717-48 (1984), or U.S. Pat. No. 5,140,103). Examples of such peptidessequences are given in Table 1 below. The conventional peptide synthesismethod may produce a mixture of peptide (with normal peptide bondedaspartic acid) and peptide analogue with isomerization of the bonding tothe aspartic acid. Generally such a mixture will be satisfactory as thepeptide will be insert in the assay. However, heating such a mixturewill normally produce isomerization of the peptide content to theiso-form.

Regarding the synthetic peptide analogues, it is possible to omit (oradd) one or more amino acid residues from (or to) the crosslinkable sitesequences without substantial loss of the ability to (a) raiseantibodies recognising the isoaspartic acid analogue of thecorresponding native collagen fragment or (b) inhibit the binding ofsuch antibodies to the said analogue of the native fragment. It ispossible to use longer collagen fragments and/or chimeric peptideanalogues to raise the antibodies and, in principle, it is not necessaryto use the same peptide analogue as the immunogen and the competitor ina competition assay.

TABLE 1

Examples of Amino Acid Sequences with Potential Sites for Cross-linkingin Various Types of Collagen to be Used as a Basis for Isoaspartic AcidContaining Synthetic Peptide Analogues According to the PresentInvention

Collagen Type 1

    ______________________________________                                        Collagen Type I                                                                   Potential sites                                                                in telopeptide analogues: N                                              ______________________________________                                                     α1 (I)                                                                     N--term.(Iso)Asp-Glu-Lys-Ser-Thr-Gly-Gly                         (SEQ ID NO: 5) (α1(I)N1)                                               α1 (I) C═term.Glu-Lys-Ala-His-(Iso)Asp-Gly-Gly-Arg                   (SEQ ID NO: 3) (α1(I)C1)                                               α2 (I) N--term.Gln-Tyr-(Iso)-Asp-Gly-Lys-Gly-Val-Gly                     (SEQ ID NO: 6) (α2)(I)N1)                                            ______________________________________                                        Collagen Type II                                                                   Potentialsites                                                             in telopeptide analogues: N                                                 ______________________________________                                                α1 (II)                                                                         N--term.  Gly-Asp-Ile-Lys-(Iso)Asp-Ile-Val                       (SEQ ID NO: 7)                                                               α1 (II) C--term. Glu-Lys-Gly-Pro-(Iso)Asp (SEQ ID NO: 8)              ______________________________________                                    

Collagen Type III

Potential sites in telopeptide analogues: N C α1 (III) N-term.(Iso)Asp-Val-Lys-Ser-Gly-Val (SEQ ID NO:9)

Preparation of Antibodies

The methods for preparation of both monoclonal and polyclonal antibodiesare well known in the art. For example, see Campbell, A. M., LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 12 (1986). It ispossible to produce antibodies to synthetic isomerized peptides byimmunisation. However, because of the relatively small molecular weightof these compounds it is preferred that the hapten be conjugated to acarrier molecule. Suitable carrier molecules include, but are notlimited to, bovine serum albumin, thyroglobulin, oval-bumin, tetanustoxoid, and keyhole limpet hemocyanin. The preferred carrier is bovineserum albumin. To present the hapten in its most immunogenic form to theantibody producing cells of the immunised animal a number of alternativecoupling protocols can be used. Suitable procedures include, but are notlimited to, glutaraldehyde, carbodiimide, and periodate. Preferredbinding agents are glutaraldehyde and carbodiimide.

The preparation of antibodies may be carried out by conventionaltechniques including immunisation with collagen fragments containingnatural isomerization or synthetic isomerized peptides conjugated to acarrier. To improve the immunogenicity it is preferred that theimmunogen be mixed with an adjuvant before injection. Examples ofadjuvants include, but are not limited to, aluminium hydroxide, Freund'sadjuvant, and immune-stimulating complexes (ISCOMs). ISCOMs can be madeaccording to the method described by Morein, B. et al., Nature308:457-460 (1984).

Either monoclonal or polyclonal antibodies to the hapten-carriermolecule can be produced. For the production of monoclonal antibodies itis preferred that mice are immunised. Spleen cells from the immunisedmouse are harvested, homogenised, and thereafter fused with cancer cellsin the presence of polyethylene glycol to produce a cell hybrid whichproduces monoclonal antibodies specific for isomerized peptide fragmentsderived from collagen. Suitable cancer cells include, but are notlimited to, myeloma, hepatoma, carcinoma, and sarcoma cells. Detaileddescriptions of the production of monoclonal antibodies are provided inGoding, J. W., in Monoclonal Antibodies: Principles and Practice,(1986). A preferred preliminary screening protocol comprises the use ofsynthetic isomerized peptides conjugated to a carrier and coated on tothe solid surface of a microtitre plate.

For the preparation of polyclonal antibodies, which are reactive withisomerized peptide fragments derived from collagen, different animalspecies can be immunised. Suitable species include, but are not limitedto, chicken, rabbit and goat. Chicken and rabbit are preferred.

Antibodies so produced may be screened for suitability for use accordingto the invention by testing for reactivity with an isoaspartic acidcontaining synthetic peptide analogue of appropriate sequence.

Antibody fragments are prepared by methods known in the art (see E.Ishikawa. Journal of Immunoassay 3:209-327 (1983)).

Conduct of Immunoassays

Accordingly, by utilisation of an immunoassay with the antibodiesprepared as above it is possible to assay a biological fluid samplewithout prior fractionation or hydrolysis. The specificity for thedesired collagen fragments in the biological fluid may be supplied bythe antibody in combination with the use of a synthetic isomerizedpeptide (against which the antibody was raised or in any event withwhich the antibody is immunochemically reactive) in the assayconstruction.

As an alternative the immunoassay may be performed using a monoclonalantibody. The basic idea of this assay design is to shift thespecificity of the assay from the antigen (synthetic peptide isomer tocollagen) to the antibody (from rabbit antiserum to monoclonalantibody). Using this construction the assay does not need to makefurther use of a synthetic peptide isomer. This version of theimmunoassay is suitably performed by incubating the patient sample or astandard solution with a peroxidase-conjugated antibody solution in amicrotiter plate precoated with purified collagenase-treated collagen.After washing, the wells of the plate are incubated in the dark with asubstrate solution. The colour reaction is stopped by the addition of astopping solution, and finally the absorbance is measured.

The immunoassays themselves may be conducted using any procedureselected from the variety of standard assay protocols generally known inthe art. As it is generally understood, the assay is constructed so asto rely on the interaction between the specific immunological bindingpartner and the desired analyte for specificity and to utilise somemeans to detect the complex formed by the analyte and the immunologicalbinding partner. The immunological binding partner may be complexed to asolid support and used as a capture immunological binding partner forthe analyte. This protocol may be run in a direct form, wherein theformation of analyte-immunological binding partner complex is detected,e.g. by a fluorescent, radioactive or enzymatic label, or it may be runin a competitive format wherein a labelled standard competes with theanalyte for the immunological binding partner. The format may also beconstructed as an agglutination assay or the complex may be precipitatedby addition of a suitable precipitant to the reaction mixture. Thespecific design of the immunoassay protocol is open to a wide variety ofchoice, and the number of clinical assay devices and protocols availablein the art is multitudinous. For a variety of such protocols, see U.S.Pat. No. 5,001,225.

The antibodies and revealing reagents for the conduct of an immunoassayusing standard detection protocols, for example radioisotope labelling,fluorescent labelling or ELISA, either in a direct or competitiveformat, may conveniently be supplied as kits which include the necessarycomponents and instructions for the assay. In one embodiment of theinvention such a kit includes a microtiter plate coated with a relevantsynthetic isomerized peptide, standard solutions for preparation ofstandard curve, a body fluid (e.g. urine) control for quality testing ofthe analytical run, rabbit antibodies reactive with the above mentionedsynthetic peptide isomer, anti-rabbit immunoglobulins conjugated toperoxidase, a substrate solution, a stopping solution, a washing bufferand an instruction manual.

Since immunoassays can be constructed using antibodies and specificsynthetic isomerized peptides, the ratios of the corresponding collagenfragment sequences in an appropriate biological fluid can be determinedas well as their individual levels and their total. Thus, the assay canbe designed to include antibodies which will result in determination ofseveral isoaspartic acid containing peptide analogues and optionally thenative peptide sequences or determination of a single isoaspartic acidcontaining peptide analogue sequence, or any desired combinationthereof.

In addition to the use of the herein specified isomerized peptides asindicators of bone resorption, bone metabolic balance is advantageouslydetermined by the substantially simultaneous determination of a markerof the formation of bone in the same or other appropriate biologicalfluid from the same individual. "Substantially simultaneous" means thesame day, preferably within 4 hours. For example such markers includeosteocalcin (also known as bone GLA protein of BGP), propeptides ofprocollagen type I, bone alkaline phosphatase and total alkalinephosphatase. Suitable methods for the determination of these markers canbe found, for example, in Delmas, P. D., et al., J. Bone Min. Res.(1986) 1:333-337.

The assay of the present invention which provides an index todetermination of the metabolic status of tissues, which generatecollagen-derived peptides and isomerized peptides when degradationoccurs, is useful in a variety of contexts. First, when considering thedegradation of type I collagen, the assays are methods to assess anabnormal condition of a subject by indicating, for example, excessivebone resorption. This may show the presence of an osteoporotic conditionor the metastatic progress of a malignancy. Other conditionscharacterised by excessive bone resorption include Paget's disease andhyperparathyroidism. Likewise, a number of other disease statesinvolving connective tissue may be monitored by determination of thedegradation of collagen. Examples are collagen type II degradationassociated with rheumatoid arthritis and osteoarthritis and collagentype III degradation in vasculitis syndrome. Since the condition of thesubject can be monitored continuously, application of these assays canalso be used to monitor the progress of therapy administered to treatthese or other conditions. Further, the assays can be used as a measureof toxicity, since the administration of toxic substances often resultsin tissue degradation.

Thus the assays may be applied in any situation wherein the metaboliccondition of collagen tissues can be used as an index of the condition,treatment, or effect of substances directly administered to the subjector to which the subject is exposed in the environment.

The following examples are intended to illustrate, but not to limit theinvention.

EXAMPLE 1

Immunoassays for Specific Peptide Sequences in Urine

Three isomerized peptides (α1(I)C1, α1(I)N1, and α2(I)N1) (see Table 1)prepared by solid-phase techniques are used for the preparation ofimmunogens. For immunisation, the peptide isomers are covalentlyattached to bovine serum albumin using carbodiimide or glutaraldehydereagents and methods well known in the art. Both monoclonal andpolyclonal antibodies are raised against the peptide isomers. Forproduction of monoclonal antibodies, Balb/c mice are immunised withpeptide isomer-BSA conjugates, and hybridoma cell lines are preparedusing standard techniques after fusion of cells from the spleen or lymphnodes with Ag8 myeloma cells. Polyclonal antibodies are raised inrabbits and chicken. Screening of both antisera and hybridoma cell mediawere performed by ELISA using microtiter plates coated with theappropriate peptide isomer-protein carrier conjugate prepared usingcarbodiimide reagents and methods well known in the art.

Assays for three of the peptide isomer sequences (α1(I) C1, α1(I)N1, andα2(I)N1) in urine are performed by an inhibition ELISA as follows:

Urine samples (10 to 25 μl) possibly containing collagen fragments orsolutions containing 0.015-15 μg peptide isomer/ml as referencestandards, respectively, are added to 75 μl of immunological bindingpartners for the peptide isomers diluted 1:5,000-1:20,000 in phosphatebuffered saline containing 0.1% Tween-20 detergent (PSB-T) and including0.1% (w/v) of BSA. Each sample is prepared in duplicate inflat-bottomed, 96-well microtiter plates previously coated with peptideisomer-protein carrier conjugate containing the appropriate peptideisomer. After 60 minutes, the plates are washed with PBS-T (3 times) andthe bound antibodies are detected by standard techniques with ahorseradish peroxidase labelled antibody prepared against the species ofthe primary antibody. Peroxidase substrate is added and the colourdevelopment is measured at 450 nm in an automated microtiter platereader after stopping the enzyme reaction using 1 M H₃ PO₄. Samplescontaining the analyte decrease the binding of primary antibody to theimmunobilised peptide isomer on the plate and thus have a reduced colourconcentration. The amount of analyte in the sample is quantified withreference to previously established curves from standards included oneach plate computed using loglin plots.

EXAMPLE 2

Assay Using a Monoclonal Antibody for Detection of Degradation Productsof Type I Collagen

Monoclonal antibodies were developed by immunisation of mice withisoaspartic acid containing α1(I)C1 synthetic peptide analogueconjugated to an appropriate carrier protein. Cell fusion, cloning andpropagation of hybridomas were performed according to standardprocedures. Screening procedures included testing of reactivity toα1(I)C1 synthetic peptide analogue immobilised on microtiter plates.

The specificity of the antibodies was tested by inhibition studies usingdifferent overlapping sequences from the C-telopeptide of type Icollagen in peptide analogue form, i.e. including the isomerized linkageas aspartic acid.

An assay using one such antibody MAbA-ISO is developed. In brief, theisoaspartic acid containing synthetic peptide analogue α1(I)C1 isconjugated to bovine serum albumin using carbodiimide or glutaraldehyde,and the conjugate is used for coating of microtiter plates. Alternativematerial can also be used, the essential feature being exposure of thesequence EKAHiDGGR (SEQ ID NO:3) sequence.

Following coating, the wells of the microtiter plates are incubated with15 μl of urine and 100 μl of MAbA-ISO conjugated to horseradishperoxidase.

After one hour the plates are washed and substrate (e.g. TMB) is added.

EXAMPLE 3

Correlation of ELISA for Isoaspartic Acid Peptide Analogue to ELISAAssays Based on Other Peptide Fragments and Antibodies

The correlation between a polyclonal ELISA according to this inventionand an MAbA7 ELISA as described in WO95/08115 on HPLC separated peptidespecies, (a) synthetic and (b) isolated from urine, was evaluated. TheMAbA7 ELISA is a competitive assay wherein the MAbA7 monoclonal raisedagainst the 8AA peptide (EKAHDGGR (SEQ ID NO:10) in which D is normalaspartic acid) is used and collagen degraded collagen immobilised in amicrotiter tray competes with collagen fragments in the sample forbinding to the monoclonal antibody (MAbA7).

The ELISAs used in this example are carried out as follows:

(a) The Polyclonal ISO-ELISA

The polyclonal ELISA is based on an immobilised synthetic peptideanalogue with an amino acid sequence of eight amino acids (8AA) specificfor an isomerised part of the C-telo-peptide of the a1-chain of type Icollagen (Glu-Lys-Ala-His-(Iso)ASP-Gly-Gly-Arg (SEQ ID NO:3)). Duringincubation with an antibody reactive with this sequence, a competitiontakes place between the immobilised peptide iso-form analogue and thebreakdown products of the α1-chain of type I collagen in the sample.

Briefly, a 25 μL sample or standard is added to each well of a formula 1antigen-coated microplate, followed by 75 μL of antiserum raised againstcollagenase treated type 1 collagen. The plates are incubated for 1 hourat room temperature under agitation and washed five times with a washingbuffer. A goat anti-rabbit immunoglobulin G horseradish peroxidaseconjugate (100 μL) is added to each well. After incubation for 1 hour atroom temperature, plates are washed five times as before. The enzymesubstrate (100 μL/well) is added, and after 15 minutes of incubation inthe dark, the reaction is stopped by adding 100 μL phosphoric acid (1mol/L). The optical density of 450 nm is measured with a microplatereader. Duplicate measurements are performed for each sample, and thedata are expressed as nanograms per mol creatinine (Cr), measured by astandard calorimetric technique. This assay is referred to herein asISO-ELISA.

The MAbA7 ELISA

MAbA7 is a monoclonal antibody raised as described above in mouseagainst the peptide of the sequence EKAHDGGR (SEQ ID NO:10), i.e. withnormal aspartic acid.

This assay version is carried out in three steps as follows:

In the first step the wells of a microtiter plate precoated withpurified collagenase-treated collagen (CTC) are incubated for 1 hour atroom temperature with 15 μl standard solution or sample and 100 μl ofperoxidase-conjugated monoclonal antibody solution.

After washing in the second step the wells are incubated in the dark for15 minutes at room temperature with 100 μl substrate solution. Finally,in the third step, the colour reaction is stopped by the addition of 100μl stopping solution. The absorbance at 450 nm is measured within 2hours.

EXAMPLE 3(a)

This example demonstrates that antibodies specific for either syntheticEKAHDGGR (SEQ ID NO:10)or EKAHiDGGR (SEQ ID NO:3) can be developed.

A mixture of synthetic EKAHDGGR (SEQ ID NO:10)and EKAHiDGGR (SEQ IDNO:3)was separated by HPLC (FIG. 1, peak 3 and peak 2, respectively).Sequence analysis confirmed that peak 2 contained isomerized aspartate.This was indicated by the sequencing operation being halted after thehistidine residue. Peak 3 contained regular aspartate and sequencing wasable to proceed normally. Fractions from the HPLC profile were collectedand tested for immunoreactivity in both MAbA7 ELISA and polyclonalISO-ELISA. It was demonstrated that the two assays detected separatepeaks, i.e. peak 2 was detected by ISO-ELISA and peak 3 by MAbA7 ELISA.

The results are shown in FIG. 2.

EXAMPLE 3(b)

This example demonstrates that ISO-ELISA-related molecules can bepurified from urine, and that these molecules can be separated intodi-peptide related species containing isomerized or regular aspartate.

First urine was immunopurified using MAbA7. MAbA7 was coupled to CNBractivated Sepharose™ according to the manufacturer's instructions. Urinewas diluted 1:3(v/v) in PBS buffer and the pH adjusted to 8.0 using 1 MNaOH. Eight-hundred ml diluted urine was recirculated on to a column (14cm³) for 24 hr at 4° C. at a flow rate of 0.8 ml/min. After washing thecolumn with 200 ml PBS buffer, pH 8.0, bound antigens were eluted using20 ml 50% saturated (NH₄)₂ SO₄ containing 1% TFA (v/v) and stored at-20° C. The eluted antigens were desalted using a 1 ml C18 Sep-Pak™column. The Sep-Pak™ column was conditioned with 20 ml 80% methanol(v/v) and equilibrated with 20 ml water containing 1% TFA (v/v) beforeadding the antigen. Bound antigens were washed with 20 ml of watercontaining 1% TFA (v/v) and eluted with 40% acetonitrile (v/v)containing 0.1% TFA (v/v), freeze-dried and stored at -20° C.

Molecules from selected peaks obtained by the above procedure werefurther separated by HPLC using trifluoroacetic acid (TFA) as ionpair.Fractions were analysed by amino acid composition, amino acidsequencing, mass spectrometry, and immunoreactivity in MAbA7 andISO-ELISA.

The fluorescence (3-hydroxypyridinium cross-links) and absorbance of theHFBA HPLC profile is demonstrated in FIG. 3. By further separation purepreparations were obtained and tested by mass spectrometry andimmunoreactivity (Table 2), as well as amino acid sequencing (Table 3)and amino acid composition (Table 4). Data demonstrates that threeseparate molecules all with the molecular weight around 2036 kDaltonscould be identified (Table 2). Amino acid sequencing was blocked afterhistidine in peak F-24-17-10, whereas the complete sequence EKAHDGGR(SEQ ID NO:10) was obtained for the two other peaks (F-26-18-09 andF-29-19-24, Table 3). Amino acid composition analysis (which does notseparate isomerised and regular aspartate) confirmed the three peakscontained the same amino acids.

These data suggest that three different, cross-linked di-peptide relatedspecies were identified in urine. The molecules differed in havingeither isomerised or regular aspartate in the sequence EKAHDGGR (SEQ IDNO:10). Peak F-24-17-10 is suggested to contain two peptide analogues ofthe sequence EKAHiDGGR (SEQ ID NO:3), peak F-26-18-09 one EKAHiDGGR (SEQID NO:3) and one EKAHDGGR (SEQ ID NO:10), and peak F-29-19-24 twocross-linked peptides of the sequence EKAHDGGR (SEQ ID NO:1).

Additionally, a similar observation was done on molecules of moleculeweight around 2039. These did not contain 3-hydroxypyridiniumcross-links (as fluorescence was absent), and the nature of thecross-linker was not determined.

                                                      TABLE 2                 

    ______________________________________                                        Detection by MAbA7 and ISO-ELISA of                                             Peptide related species separated by HPLC from Urine                                                                  ISO-                                  Fraction    MAbA7 ELISA                                                       No. Mass Pyr DPyr ng/ml ng/ml                                               ______________________________________                                        F-18-17-14 2038.2                 58    974                                     F-20-18-18            2039.5                         406      350                                                    F-22-18,19,20-16      2038.9                                                                     972      118                                               F-24-17-10            2036.1                                                 +                    114     2110       F-26-18-09            2036.2     +                   2114     2203                                                   F-26-18-12            1858.9                                                            +          85      783       F-29-19-24            2036.0     +                   3149      221                                                   F-30-20-12            1858.8                                                           +        2108               ______________________________________                                                                                33                                

                                                            TABLE 3           

    ______________________________________                                                  Sequence Data for Peptide related molecules in                        F-24-17-10,  F-26-18-09, and F-29-19-24                                               F-24-17-10 F-26-18-09                                                                             F-29-19-24                                                                             Expected                               ______________________________________                                        1     Glu        Glu        Glu      Glu                                        2 --  -- -- --                                                                3        Ala         Ala         Ala         Ala                              4        His         His         His         His                              5      --                Asp         Asp         Asp                          6 --    Gly         Gly         Gly                                           7    --    Gly         Gly         Gly                                        8               --       Arg         Arg         Arg                          9      -- -- -- --                                                            10         -- -- -- --                                                      ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Amino Acid Composition of the Peptides                                          F-24-17-10, F-26-18-09,  and F-29-19-24 (residue/peptide)                               F-24-17-10 F-26-18-09                                                                            F-29-19-24                                                                             Expected                              ______________________________________                                        Asp     1.0        1.0           0.9  1                                         Glu         1.3          1.2         1.3 --                                   Ser         0.4          0.2         0.6    --                                Gly         2.2          2.0         2.4          2                           His         1.4          1.0         1.0          1                           Thr         0.1         0.0        0.2        --                              Ala         1.0          1.0         1.0          1                           Pro         0.0          0.0         0.0       --                             Tyr         0.0          0.0         0.0     --                               Val         0.1          0.1         0.1          --                          Met         0.0          0.0         0.0        --                            Ile         0.1          0.1         0.1      --                              Leu         0.1          0.1         0.2           --                         Lys         0.3          0.1         0.3--                                    Arg        0.8          0.9         0.8          1                            Total        8.8          7.6         9.0          7                        ______________________________________                                    

The polyclonal assay is shown to be specific for the peptide analoguecontaining isoaspartic acid residues and the MAbA7 assay is specific forthe peptide containing normal aspartic acid residues. Di-peptideanalogues containing both the isomerised as well as the non-isomerisedaspartic acid will be detected by both assays.

The application of these two assays to a urine sample therefore allows acomparison to be made of the level of isoaspartic acid containingisomerized peptide and the level of the normal form, giving anindication of the degree to which old established bone tissue is beingbroken down.

EXAMPLE 4

The clinical significance of the presence of isomerised peptideanalogues in serum was investigated by subjecting serum samples frompostmenopausal women taken before and after treatment with abisphosphonate, Ibandronate (Boehringer Mannheim), to analysis in both aserum version of the ISOELISA polyclonal assay described above and theMabA7 based ELISA monoclonal assay.

The assay developed for measurement of type I degradation products inserum is based on an immobilised synthetic peptide/peptide isomermixture with the amino acid sequence EKAHDGGR (SEQ ID NO:10) present inpeptide and in peptide isomer form. Rabbits were immunised with theisomerized peptide conjugated to BSA using a two step carbodiimideprocedure. If the isomerized peptide is substantially more antigenic inthe rabbits employed than the peptide form of the amino acid sequenceone can immunise with the peptide/peptide isomer mixture conjugated toBSA. For coating of microtitre plates the peptide was conjugated tothyroglobulin using glutaraldehyde. During incubation with this antibodya competition takes place between the immobilised peptide isomer (theimmobilised peptide being inactive in the assay) and the breakdownproducts of type I collagen in serum. As the content of the peptide inthe solution increases, less antibody will bind to the immobilisedpeptide isomer leading to a decreasing optical density.

In brief, 50 μl Standard (synthetic 8AA-peptide isomer/peptide mixture)or unknown sample in a test tube is added 100 μl Assay Buffer (500 mMTRIS, 0.0%. Tween 20, 1.0% BSA; pH=6.5). From these 150 μl, 50 μl ispipetted into the appropriate wells in the pre-coated ELISA plate. Then50 μl Antibody Solution (rabbit antiserum to EKAHiDGGR (SEQ ID NO:3)diluted 1+20.000 in Assay Buffer) is added to each well, the plate iscovered with sealing tape and incubated at room temperature for 60 minon a shaking device. All the following procedures were also carried outat room temperature. After incubation the plates were washed three timeswith diluted Washing Buffer (25 mmol/l TRIS and 50 mmol/l NaCl pH=7.2).

Peroxidase conjugated Antibody (HRP-conjugated goat antibodies to rabbitIgG (Jackson Immunochemicals, PA) diluted 1+4000 in Assay Buffer), 100μl/well) was added and the sealed wells were incubated 60 min on ashaking device. Following another washing procedure, 100 μl of TMBSubstrate Solution was added to all wells which were sealed andincubated for 15 min. The enzyme reaction was stopped after 15 min byaddition of 100 μl Stopping Solution. The optical density was read in anELISA-reader at 450 nm.

A calibration curve was constructed on a log-linear graph paper byplotting the mean absorbances of the five standards (25 ng/ml-500ng/ml). The concentration of EKAHiDGGR (SEQ ID NO:3) equivalent in eachpatient specimen were determined by interpolation on the calibrationcurve.

The results are shown in FIGS. 4 and 5. In FIG. 4, no significant changeis seen in the serum level of molecules reactive in the monoclonal assaydirected to molecules competing for antibody reactive with the peptidesequence EKAHDGGR (SEQ ID NO:10) before and after fifteen monthstreatment with a therapeutic expected to reduce the rate of boneresorption. In FIG. 5, one sees a drop of more than 60% followingtreatment for six months with 2.5 mg/day of Ibandronate in the level ofmolecules reactive in the polyclonal assay directed to moleculescompeting with the non-peptide EKAHiDGGR (SEQ ID NO:3) for antibody inthe polyclonal serum, reflecting the expected result of the therapeutictreatment.

This example indicates that the sum of peptides in serum incorporatingthe sequence EKAHDGGR (SEQ ID NO:10), which may include cross-linkedpeptides of the kind described in U.S. Pat. No. 5,455,179 and similarmolecules based on other cross-links or not cross-linked at all, are notindicators of the rate of bone resorption, but that the relatednon-peptide molecules produced by isomerisation of the bonding of theaspartic acid in the amino acid sequence are.

It is suggested that the specificity for the isomerised analogue of theoctapeptide leaves the ISOELISA insensitive to molecules present inserum which are reactive in the MabA7 ELISA. The level of isomerisationis believed to be related to the age of the bone, being therefore aquantifiable marker for turnover of mature bone, whilst the moleculesinterfering in the monoclonal assay do not relate to bone resorption andcould originate from the turnover of "young" collagen molecules, e.g.from proteolytic breakdown of extracellular collagen not yetincorporated into the bone matrix, or generated during turnover ofshort-lived type 1 collagen at non-skeletal sites.

Furthermore, although it is believed that the body has mechanisms forrepairing damage to proteins by isomerisation at aspartate, it may bethat in bone collagen is shielded from these repair mechanisms makingisomerisation an unusually good indicator of the resorption of "old"collagen in the case of bone.

Whilst the invention has been described with reference to specificexamples, many variations thereof are possible within the scope of theinvention. For instance the invention may be applied to the asparticacid or asparagine containing proteins generally and not only tocollagens.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 10                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 2                                                               (D) OTHER INFORMATION: - #Pyridinoline or deoxypyridinoline                       cross-link - #to lysine residue at position 2 of identical                    second ch - #ain.                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Glu Lys Ala His Asp Gly Gly Arg                                           1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 2                                                               (D) OTHER INFORMATION: - #Pyridinoline or deoxypyridinoline                        cross-link - #to lysine residue at position 2 of identical                    second ch - #ain.                                                        (A) NAME/KEY: Other                                                           (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #Aspartic Acid or Beta-Aspartic Acid        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Glu Lys Ala His Xaa Gly Gly Arg                                           1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #Beta-Aspartic Acid                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Glu Lys Ala His Xaa Gly Gly Arg                                           1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #Aspartic Acid or Beta-Aspartic Acid        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Glu Lys Ala His Xaa Gly Gly Arg                                           1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #Beta-Aspartic Acid                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Xaa Glu Lys Ser Thr Gly Gly                                               1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #Beta-Aspartic Acid                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Gln Tyr Xaa Gly Lys Gly Val Gly                                           1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #Beta-Aspartic Acid                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Gly Asp Ile Lys Xaa Ile Val                                               1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #Beta-Aspartic Acid                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Glu Lys Gly Pro Xaa                                                       1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #Beta-Aspartic Acid                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Xaa Val Lys Ser Gly Val                                                   1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Glu Lys Ala His Asp Gly Gly Arg                                           1               5                                                           __________________________________________________________________________

What is claimed is:
 1. A method of measuring the rate of degradation oftype I collagen comprising determining the amount in a body fluid of oneor more fragments of type I collagen having an amino acid sequencecomprising isoaspartic acid.
 2. The method of claim 1, wherein thefragments comprise the amino acid sequence EKAHiDGGR (SEQ ID NO:3). 3.The method of claim 1, comprising determining the amount present in saidbody fluid of: (a) the peptide of formula 2 (SEQ ID NO:2), below:##STR4## wherein K-K-K is any naturally occurring cross-link and * isisoaspartic acid, or (b) one or more peptide analogues containing anepitope present in the peptide of formula 2 which contains isoasparticacid.
 4. The method of claim 3, wherein the cross-link is pyridinolineor deoxypyridinoline.
 5. The method of claim 1, wherein saiddetermination is carried out using an immunological binding partnerwhich specifically binds to said amino acid sequence comprisingisoaspartic acid.
 6. The method of claim 5, wherein the immunologicalbinding partner is an antibody, or an active fragment thereof, raisedagainst an amino acid sequence of type I collagen comprising anisoaspartic acid residue.
 7. The method of claim 5 or 6, wherein theimmunological binding partner is contacted with a synthetic peptidecomprising said amino acid sequence containing isoaspartic acid.
 8. Themethod of claim 2, 3, or 4, wherein said determination is carried outusing an immunological binding partner which specifically binds to theamino acid sequence EKAHiDGGR (SEQ ID NO:3).
 9. The method of claim 8,wherein the immunological binding partner is an antibody, or an activefragment thereof, raised against an amino acid sequence of type Icollagen comprising the amino acid sequence EKAHiDGGR (SEQ ID NO:3). 10.The method of claim 8, wherein the immunological binding partner iscontacted with a synthetic peptide comprising the amino acid sequenceEKAHiDGGR (SEQ ID NO:3).
 11. A method of obtaining information regardingtype I collagen degradation in a patient, comprising measuring in a bodyfluid the relative amounts of: (a) at least one aspartic acid containingpeptide derived from type I collagen and (b) a corresponding isoasparticacid containing peptide analogue.
 12. The method of claim 11, whereinthe isoaspartic acid containing peptide comprises the amino acidsequence EKAHiDGGR (SEQ ID NO:3).
 13. An immunological binding partnerwhich specifically binds to an amino acid sequence of type I collagencontaining an isoaspartic acid residue.
 14. The immunological bindingpartner of claim 13, which specifically binds to the amino acid sequenceEKAHiDGGR (SEQ ID NO:3) or to an epitope included in said sequence andcontaining iD.
 15. The immunological binding partner of claim 13 or 14which is a monoclonal antibody or an active fragment thereof.
 16. Theimmunological binding partner claim 13 or 14 coupled to a detectablemarker.
 17. The immunological binding partner of claim 15 coupled to adetectable marker.
 18. A cell line producing the monoclonal antibody ofclaim
 15. 19. A synthetic peptide having an amino acid sequence of typeI collagen which contains an isoaspartic acid residue.
 20. The syntheticpeptide analogue of claim 19, wherein there is a glycine residueadjacent the isoaspartic acid residue.
 21. The synthetic peptide ofclaim 20, comprising the amino acid sequence EKAHiDGGR (SEQ ID NO:3).22. A test kit for measuring type I collagen degradation, comprising theimmunological binding partner of claim 13 or
 14. 23. A test kit formeasuring type I collagen degradation, comprising the monoclonalantibody or active fragment of claim 15.